血清has_circ_0000400在结肠癌中的表达及生物学功能研究

Expression and biological function of serum has_circ_0000400 in colon cancer

目的分析血清has_circ_0000400在结肠癌中的表达水平及生物学功能,通过检测结肠癌患者血清has_circ_0000400表达量,通过生物信息学方法分析其作用机制,探讨has_circ_0000400作为结肠癌肿瘤标志物的可能性。方法选取2018年1月至2020年10月常熟市第二人民医院普外科收治的78例结肠癌患者作为结肠癌组,另随机选取2020年9月于常熟市古里人民医院参加健康体检的78例健康体检者作为对照组。两组均采用实时定量反转录聚合酶链反应技术检测血清中has_circ_0000400相对表达水平,并结合患者相关临床病理特征综合分析,培养结肠癌标准细胞株SW480、LOVO、HT-29与正常人结肠黏膜上皮细胞株HCoEpiC对结果验证。通过生物信息学软件预测has_circ_0000400的靶向基因,并进一步预测miRNA的靶向蛋白。采用受试者工作特征曲线(receiver operating characteristic curve,ROC曲线)分析血清has_circ_0000400、糖类抗原19-9(carbohydrate antigen 19-9,CA19-9)及联合检测对结肠癌的诊断价值。结果结肠癌组血清has_circ_0000400相对表达量、CA19-9水平均高于对照组,差异有统计学意义(P<0.05)。不同分化程度、TNM分期及有无淋巴结转移患者血清has_circ_0000400表达量比较差异有统计学意义(P<0.05)。平板克隆形成实验以接种100个细胞为模型,培养1周后,沉默has_circ_0000400后的SW480细胞的克隆形成率为54.28%,正常SW480细胞的克隆形成率为21.44%,侵袭实验显示沉默SW480细胞后结肠癌细胞侵袭能力显著下降。蛋白质免疫印记实验显示,has_circ_0000400负调控EBF3,侵袭实验显示过表达EBF3后,细胞增殖能力与侵袭能力显著下降。CCK8增殖实验显示,过表达EBF3的SW480细胞增殖能力显著下降。结论血清hsa_circ_0000400可作为结肠癌新的参考标志物,与CA19-9联合检测诊断结肠癌更为准确,但其具体分子机制仍需进一步探索。

Objective To analyze the expression level and biological function of serum has_circ_0000400 in colon cancer,analyze its mecha-nism of action by bioinformatics methods,and to explore the possibility of has_circ_0000400 as a tumor marker colon cancer.Methods 78 colon cancer patients admitted to the Department of General Surgery of the Second People's Hospital of Changshu City from January 2018 to October 2020 were selected as the colon cancer group,and another 78 healthy examination subjects who participated in health checkups at Changshu Guli People's Hospital,in September 2020 were randomly selected as the control group.Both groups used real-time quantitative reverse transcription polymerase chain reaction technology to detect the relative expression level of has_circ_0000400 in serum,and combined with the relevant clinico-pathological features of patients to comprehensively analyze the results,and cultured colon cancer standard cell lines SW480,LOVO,and HT-29 with the normal human colonic mucosal epithelial cell line,HCoEpiC,to verify the results.The target genes of has_circ_0000400 were predicted by bioinformatics software,and the target proteins of miRNA were further predicted.Subject operating characteristic curve(ROC curve)was used to analyze the diagnostic value of serum has_circ_0000400,carbohydrate antigen 19-9(CA19-9),and the combined examination for colon cancer.Results The relative expression level of serum has_circ_0000400 and CA19-9 in the colon cancer group were higher than those in the control group,and the differences were statistically significant(P<0.05).There were significant differences in the expression of serum has_circ_0000400 in patients with different differentiation degree,TNM stage and lymph node metastasis(P<0.05).Plate clone formation assay was modeled by inoculat-ing 100 cells,and after 1 week of culture,the clone formation rate of SW480 cells after silencing has_circ_0000400 was 54.28%,and that of control SW480 cells was 21.44%,and the invasion assay showed that the invasive ability of colon cancer cells was significantly decreased after silencing SW480 cells.Protein immunoblotting assay showed that has_circ_0000400 negatively regulated EBF3,invasion assay showed that overexpression of EBF3 resulted in a significant decrease in the proliferative and invasive capacity of the cells.CCK8 proliferation assay showed that the proliferative capacity of SW480 cells overexpressing EBF3 was significantly decreased.Conclusion Serum hsa_circ_0000400 can be used as new reference marker for colon cancer,and are more accurate when tested in combination with CA19-9 for colon cancer,but its specific molecular mechanism still needs to be further explored.